Interaction Between Lrrc10 And Srf, And Cell Function Study Of Nulp1

Heart diseases have become the most serious diseases that threaten human beings. It is a critical prerequisite to understand the normal progress of heart development and the mechanisms underlying this kind of diseases occur.The human Lrrc10 gene has been cloned previously by our lab and its expression analysis in various organs of adult mouse and human embryos by Western blot proved that Lrrc10 is a cardiac-specific expression factor. Co-immunoprecipitation and GST Pull-Down show that Lrrc10 could interact with the Srf gene, and it has also been validated that Lrrc10 inhibits cardiac hypertrophy by inhibiting the activation of Srf on the ANF. To further investigate how Lrrc10 interacts with Srf, Lrrc10 and Srf have been subcloned, seperately, based on their domain structures. Immunoprecipitation, PULL-DOWN and report gene analyses have been used with each subclones and the results indicate that Lrrc10 interacts with Srf though the conservative Lrr domain unit, and Srf interacts with Lrrc10 through the MADS domain. LRRC10 polyantibody was prepared to further explore the role of Lrrc10 in cardiac hypertrophy. RT-PCR and Western Blot result show that Lrrc10 expression is reduced in ISO stimulatied pathological hypertrophy mice and cardiac hypertrophy human samples.It has been previously found by our lab that the Nulpl Gene inhibits the P19CL6 cell cardiomyocyte differentiation through Wnt/β-catenin pathway. To further investigate the function of Nulpl in cardiac development, subcellular localization has been usedand the results indicated that Nulpl could interact withβ-atenin. Western blot also showed that Nulpl expression was inhibited in the 6th day of P19 cardiomyocyte differentiation, consistent with the previous RT-PCR results. Furthermore, we established two stable P19 cell lines, the Nulpl overexpression line of P19 cells, called P19-Nulpl line, and the Nulpl knock-down line of p19 cells, called P19-Nulp1-I line. Western blot analyses indicated that the rate of cardiomyocyte differentiation was inhibited in P19-Nulpl line and increased in P19-Nulp1-I line than those of control. These results further confirmed that Nulpl Inhibits the Cardiomyocyte Differentiation through canonical Wnt signaling.